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Release of a ubiquitin brake activates OsCERK1-triggered immunity in rice

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Abstract

Plant pattern-recognition receptors perceive microorganism-associated molecular patterns to activate immune signalling1,2. Activation of the pattern-recognition receptor kinase CERK1 is essential for immunity, but tight inhibition of receptor kinases in the absence of pathogen is crucial to prevent autoimmunity3,4. Here we find that the U-box ubiquitin E3 ligase OsCIE1 acts as a molecular brake to inhibit OsCERK1 in rice. During homeostasis, OsCIE1 ubiquitinates OsCERK1, reducing its kinase activity. In the presence of the microorganism-associated molecular pattern chitin, active OsCERK1 phosphorylates OsCIE1 and blocks its E3 ligase activity, thus releasing the brake and promoting immunity. Phosphorylation of a serine within the U-box of OsCIE1 prevents its interaction with E2 ubiquitin-conjugating enzymes and serves as a phosphorylation switch. This phosphorylation site is conserved in E3 ligases from plants to animals. Our work identifies a ligand-released brake that enables dynamic immune regulation.

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Fig. 1: OsCIE1 interacts with OsCERK1 and negatively regulates OsCERK1-mediated immunity.
Fig. 2: OsCIE1 ubiquitinates and suppresses OsCERK1’s kinase activity.
Fig. 3: OsCERK1 phosphorylates and blocks OsCIE1’s ubiquitination activity promoting immunity.
Fig. 4: A phosphorylation switch and the brake/release model for OsCIE1-OsCERK1-mediated immunity.

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Data availability

All data are available within this Article and its Supplementary Information. Original gel blots are shown in Supplementary Information Fig. 1. Original data points in graphs are shown in the Source Data files. These data were also uploaded on the Figshare (https://doi.org/10.6084/m9.figshare.21972446). DrCHIP-U-box/UbcH5a structure (PDB: 2OXQ), and HsCHIP-U-box/UBC13 (PDB: 2C2V) were obtained from the Protein Data Bank (PDB). Coordinates have been deposited in PDB under the accession code 7XED (OsCIE1-U-box/OsUBC8, https://www.rcsb.org/structure/7XED), 8HQB (OsCIE1-U-box, https://www.rcsb.org/structure/8HQB) and 8HPB (OsCIE1-U-boxS237D, https://www.rcsb.org/structure/8HPB). The chemical shift data were deposited at Biological Magnetic Resonance Data Bank with the accession code 36528 (OsCIE1-U-box), 36527 (OsCIE1-U-boxS237D). Validation reports for the crystal structure of the OsUbox and OsUBC8 complex, NMR structure of OsUboxWT and NMR structure of OsUboxS237D are provided in Supplementary Tables  5,  6 and 7, respectively. Source data are provided with this paper.

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Acknowledgements

We sincerely thank the staff at beamline BL02U1 of Shanghai Synchrotron Radiation Facility for assistance during data collection; the staff members of the National Facility for Protein Science in Shanghai, China for providing technical support and assistance in NMR data collection; and Z. Yang of the Chemical Biology Core Facility at the Shanghai Institute of Biochemistry and Cell Biology for assistance on SPR experiments. We gratefully thank D. Lv (Shanghai Jiao Tong University), J. Liu, C. Wang and H. Yang (Center for Excellence in Molecular Plant Sciences, CEMPS) for help with biochemical experiment guidances; Y. Deng, Z. He, J. Liu, K. Zhai, B. Yan, J. Tang, X. Gong and L. Huang (CEMPS) for guidance for rice pathogen inoculation, rice planting and informative discussions; L. Wan (CEMPS) and Q. Zhang (East China Normal University) for help with Arabidopsis mutants ordering and transforming; J. Chu (CEMPS) for help with Arabidopsis planting; and C. Ma and W. Cai (CEMPS) for help with confocal imaging. This research was supported by the National Science Foundation (32088102, 32001886, 31825003, and 31870218), CAS Project for Young Scientists in Basic Research (YSBR-011), the Strategic Priority Research Program “Molecular Mechanism of Plant Growth and Development’’ of the Chinese Academy of Sciences (XDB0630103), National Key R&D Program of China (2019YFA0904703), the Basic Research Zone Program of Shanghai (JCYJ-SHFY-2022-012), the National Key Research and Development Program of China (2023YFF1000300, 2023YFF1000302 and 2023YFF1000303), and Life Science Editors for editing assistance. We thank Tanon Science & Technology (Shanghai, China) for experiment devices support. E.W. acknowledges support from the XPLORER PRIZE.

Author information

Author notes

  1. These authors contributed equally: Gang Wang, Xi Chen, Chengzhi Yu

Authors and Affiliations

  1. National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China

    Gang Wang, Xi Chen, Chengzhi Yu, Xiaobao Shi, Wenxian Lan, Chaofeng Gao, Jun Yang, Huiling Dai, Xiaowei Zhang, Zuhua He, Yu Zhang & Ertao Wang

  2. The New Cornerstone Science Laboratory, Shenzhen, China

    Gang Wang, Xi Chen, Xiaobao Shi, Chaofeng Gao, Jun Yang, Huiling Dai, Xiaowei Zhang, Boyu Zhao & Ertao Wang

  3. University of the Chinese Academy of Sciences, Beijing, China

    Xi Chen, Chengzhi Yu, Xiaobao Shi & Chaofeng Gao

  4. National Engineering Research Center for Sugarcane, Fujian Agriculture and Forestry University, Fuzhou, China

    Huili Zhang

  5. School of Life Science, Shanghai Normal University, Shanghai, China

    Boyu Zhao & Nan Yu

  6. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Qi Xie

  7. School of Life Science and Technology, Shanghai Tech University, Shanghai, China

    Zuhua He & Ertao Wang

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Contributions

G.W. and E.W. conceived and designed the experiments. G.W. and X.C. performed most experiments, including the pathoassays, agronomic traits analysis, Y2H, SLC, co-IP, RNA analysis, in vivo degradation assay, in vivo/in vitro phosphorylation & ubiquitination assay, ROS detection, membrane fraction, MAPK detection and western blots. C.Y. carried out the crystallization, structure determination and size-exclusion chromatography assays supervised by Y.Z.. X. S. assisted to do pathoassays. C. G. performed the subcellular localization. Q.X. provided ubiquitination associated vector & system and gave the significant suggestion to rescue this project when we were in dilemma. W.L. determined NMR structures. Z.H.’s lab enabled our rice planting in paddy field and the pathogen inoculation assays. Other authors contributed to the analytical, molecular cloning, and transformation work. G.W., X.C., C.Y., Y.Z. and E.W. analysed the data. C.Y. and Y.Z. modified the crystallization part of the manuscript. G.W. and E.W. wrote the paper.

Corresponding authors

Correspondence to Zuhua He, Yu Zhang or Ertao Wang.

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Nature thanks Yoji Kawano and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer review reports are available.

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Extended data figures and tables

Extended Data Fig. 1 OsCERK1 overexpression enhances disease resistance with a fitness cost.

a, Relative expression of OsCERK1. The rice CYCLOPHILIN2 gene served as an internal control. (Values are means ± SD, n = 3, biologically independent samples). b-d, Rice blast disease resistance against M. oryzae (TH12) of OsCERK1-mCherry overexpression lines. b, Leaves were photographed at 7 dpi with punch injection inoculation in the leave sheath of 1.5-month rice plants in the paddy field. Scale bars, 1 cm. c, Lesion area, values are means ± SD (n = 15 leaves from different rice plants). d, Relative fungal growth, values are means ± SD (n = 3, biologically independent samples). e, Bacterial blight disease resistance of OsCERK1-mCherry overexpression lines, 14 dpi with Xoo strain PXO99A. Scale bars, 1 cm. Lesion length were measured. Values are means ± SD (n = 88). f, Mature plants of NIPB and the OsCERK1-mCherry overexpression transgenic line (OEOsCERK1-mCherry). Scale bars, 10 cm. g-j, Main panicle grain filling rate (single triangle means the average grain filling rate of five main panicles per plant) (g), Single main panicle grain yield per plant (single triangle means the average grain yield of five main panicles per plant) (h), Total grain yield per plant (i), and thousand grain yield per plant (j) were significantly lower in OEOsCERK1-mCherry transgenic plants. Values are means ± SD (n = 20 rice plants). All the data above were analyzed by one-way ANOVA with Tukey’s test. Different letters indicate statistical significance (P < 0.05). All the experiments above were repeated three times with similar results.

Source Data

Extended Data Fig. 2 OsCIE1 interacts with OsCERK1.

a, Phylogenetic tree of OsCIE1 and related U-box E3 ligase homologs from rice and Arabidopsis. The evolutionary history was inferred using the Neighbor-Joining method using ClustalX2 (version 2.1) and MEGA X. The bootstrap values (1,000 replicates) are indicated. The length of the branch is proportional to the degree of divergence. The scale bar represents the average number of nucleotide replacements per site. b, In vitro pull-down assay shows the interactions of OsCERK1ic with OsCIE1. The purified GST, GST-OsCIE1 proteins were incubated with MBP or MBP-OsCERK1ic. GST-pulldown was performed with glutathione resin and western blots probed with GST and MBP antibodies. Coomassie Brilliant Blue (CBB) staining was employed to visualize the entire input, revealing the loading of each protein. c, Subcellular co-localization of the GFP-OsCIE1, free GFP, and OsCERK1-mCherry /ER marker (The full-length cDNA of the AAPT2 gene (AT3G25585)) in rice protoplasts. Scale bars, 10 μm. d, A split-luciferase complementation assay shows the interaction of OsCERK1 and OsCIE1 in N. benthamiana leaves. Leaves were imaged 3 days after transient transformation. e, Co-immunoprecipitation assays show interactions of OsCERK1 with OsCIE1. Wild-type rice protoplasts transiently expressing OsCERK1-FLAG and OsCIE1-HA were incubated for 17 h. Total proteins were extracted and immunoprecipitated with FLAG beads and then detected with anti-HA antibody. All the experiments above were repeated three times with similar results.

Extended Data Fig. 3 Oscie1 mutant shows enhanced immunity responses compared with wild-type NIPB.

a, Left: CRISPR–Cas9-mediated mutations in the 3rd exon of the OsCIE1 gene (exons indicated by blue boxes) in the wild-type NIPB rice used in this study and these mutations do not affect the expression of OsCIE1 but are expected to create truncated OsCIE1 protein. The underlined sequence in the wild-type (WT) indicates the region targeted by sgRNA. The number 709 indicates the nucleotide position in the OsCIE1 coding sequence. +1 and −1 indicate frame shifts in the mutant lines. Right: No significant difference of the transcript levels of OsCIE1 was detected between wild-type NIPB and Oscie1 mutants. CYCLOPHILIN2 was used as internal control for normalization. Values are means ± SD (n = 3, biologically independent samples). b, Two independent Oscie1 CRISPR–Cas9 transgenic mutant lines display an elevated ROS burst upon treatment with 1 × 10−6 M chitin for 1 h. Values are mean relative light units (RLU) ± SD (n = 8 seedlings). c, Transcript levels of plasma membrane NADPH oxidase gene, OsRbohH (LOC_Os12g35610) and catalase gene OsCATA (LOC_Os02g02400) was respectively increased and reduced before and after 1 × 10−6 M chitin treatment for 2 h in Oscie1 mutants’ stems compared with wild-type. CYCLOPHILIN2 was used as internal control for normalization. Values are means ± SD (n = 3, biologically independent samples). d, e, Bacterial blight lesion lengths of wild-type, Oscie1 mutants, OsCIE1 complementation lines, Oscerk1-1, and Oscerk1-1/Oscie1-1 double mutant plants inoculated with PXO99A. d, Lesions were photographed at 14 dpi. Scale bars, 1 cm. e, Average values of lesion length are means ± SD (NIPB, n = 99; Oscie1-1, n = 92; Oscie1-2, n = 65; Oscie1-1COMWT1, n = 39; Oscie1-1COMWT2, n = 92; Oscerk1-1, n = 102; Oscerk1-1/Oscie1-1, n = 216). f, Oscie1 and spl11 mutants showed lower mycorrhizal colonization compared to wild-type. After inoculation with R. irregularis for 8 weeks, rice roots were harvested, rinsed, and stained with ink-acetic acid. AM colonization was quantified by grid line intersect method40. Values are means ± SD (n = 10, biologically independent samples). g, Oscie1-1 complemented with expression of wild-type OsCIE1 genomic DNA (the same as c-e) have nearly wild-type levels of ROS burst when treated with 1 × 10−6 M chitin for 1 h. Values are mean relative light units (RLU) ± SD (n = 8 seedlings). Data above were analyzed by one-way ANOVA with Tukey’s test. Different letters indicate statistical significance (P < 0.05). Experiments (b–g) were repeated three times with similar results.

Source Data

Extended Data Fig. 4 OsCIE1 and homologs negatively regulates OsCERK1 to balance immunity and yield.

a, Mature plants of wild-type, Oscie1-1, Oscie1-2, Oscerk1-1 and Oscerk1-1/Oscie1-1 transgenic line. Scale bars, 10 cm. b-e, Main panicle grain filling rate per plant (single triangle means the average grain filling rate of five main panicles per plant) (b), Single main panicle grain yield per plant (single triangle means the average grain yield of five main panicles per plant) (c), Total grain yield per plant (d), and thousand grain yield per plant (e) were significantly decreased in Oscie1 mutant plants, which can be largely restored in Oscerk1-1/Oscie1-1 double mutant plants. Values are mean ± SD (n = 20 rice plants). f, Introgression of the Oscerk1-1 mutation allele into Oscie1-1 by crossing eliminates ROS generation in these plants mimicking Oscerk1-1 upon treatment with 1 × 10−6 M chitin for 1 h. Values are mean relative light units (RLU) ± SD (n = 8 seedlings). g, OsPUB9, OsPUB10 and SPL11 interact with OsCERK1 in yeast two-hybrid assays, whereas OsPUB3 does not. h, CRISPR–Cas9-mediated mutations in the 1st exon of the SPL11 gene (exons indicated by blue boxes) in the wild-type NIPB rice used in this study. The underlined sequence in the wild-type indicates the region targeted by sgRNA. The number 288 and 261 indicates the nucleotide position in the SPL11 coding sequence. -5 and −64 indicate frame shifts in the mutant lines. i, Rice plants with mutations of SPL11 and OsCIE1 (spl11/Oscie1-1 double mutants) display exacerbated cell death and ROS burst. Plants were grown in greenhouse and leaves were photographed about 1 month after seedling transplanting. DAB staining indicates H2O2 accumulation, and NBT superoxide. For b-f, the data were analyzed by one-way ANOVA with Tukey’s j(a-g, i) were repeated three times with similar results.

Source Data

Extended Data Fig. 5 OsCIE1 can poly-ubiquitinate kinase active OsCERK1 in vitro.

a, OsCIE1 exhibited E3 ubiquitin ligase activity. GST-OsCIE1 and GST-OsCIE1C234A, W261A fusion proteins were assayed for E3 activity in the presence of E1, E2, and HA tag ubiquitin (HA-Ub). After reaction for 1 h, ubiquitin was detected by western blot using an anti-HA antibody (Anti-HA), and auto-ubiquitination of GST-OsCIE1 were detected by anti-GST antibody (Anti-GST). “m” refers to the OsCIE1 U-box mutant (OsCIE1C234A, W261A) as a negative control. b, OsCIE1 poly-ubiquitinated OsCERK1 in vitro. The K0 mutant ubiquitin (K0Ub) in which seven lysines were replaced with arginine diminished the ladder of ubiquitinated OsCERK1. Membranes were imaged after a long exposure. c, Yeast two-hybrid assays show that kinase dead mutation of OsCERK1 dramatically dampened its interaction with OsCIE1. d, The in vitro pull-down assay reveals much stronger interactions between OsCIE1 and wild-type OsCERK1ic versus the kinase-dead form. The purified GST, GST-OsCIE1 proteins were incubated with MBP, MBP-OsCERK1ic or two kinase-dead versions of MBP-OsCERK1ic (K351E or T484A). GST-pulldown was performed with glutathione resin and western blots probed with GST and MBP antibodies. e, GST-OsCIE1 ubiquitinated wild-type MBP-OsCERK1ic-FLAG but not the two kinds of kinase-dead mutation (MBP-OsCERK1icK351E-FLAG or MBP-OsCERK1icT484A-FLAG). f, OsCIE1 poly-ubiquitinated OsCERK1 but not MBP-tagged OsFLS2 intracellular domain (OsFLS2ic), OsRac1, OsHIR1, and OsMAPK6 in vitro. Grey arrows indicated the protein bands without ubiquitin modification. All the immunoblotting was individually conducted on separated gels in parallel and all the experiments above were repeated three times with similar results.

Extended Data Fig. 6 SPL11 and OsCIE1 specifically interact with and ubiquitinate OsCERK1.

a,b, Yeast two-hybrid analysis with intracellular domains of all the OsLysM-RLKs (OsRLK1-10 including OsCERK1 or empty vector) fused to the DNA binding domain (GBK) and SPL11 (a)/OsCIE1 (b) constructs fused to the Activation domain (GAD) as indicated. Yeast was grown on -2SD (-Leu-Trp) or -3SD supplied with 3 mM 3-AT (-His-Leu-Trp +3 mM 3-AT) medium. c, Yeast two-hybrid analysis with full length of OsLysM-RLPs including CEBiP and LYP4/6 fused to the DNA binding domain (GBK) and SPL11/OsCIE1 constructs fused to the Activation domain (GAD) as indicated. Yeast was grown on -2SD (-Leu-Trp) or -3SD supplied with 3 mM 3-AT (-His-Leu-Trp +3 mM 3-AT) medium. d, In vitro ubiquitination of MBP tagged OsFLS2, OsRLK5, OsRLK7 and OsCERK1 intracellular domains with 3 × FLAG fused to their C terminals by OsCIE1 or SPL11. OsFLS2 and OsRLK5 served as negative controls. Reactions were incubated at 30 °C for 1 h. The ubiquitination of these substrates by SPL11 or OsCIE1 was detected by immunoblotting with the anti-Flag antibody (Anti-FLAG). e, The OsCIE1 homologs SPL11 and OsPUB9 also ubiquitinated OsCERK1, and OsPUB10 did not. Experiments above were repeated three times with similar results.

Extended Data Fig. 7 Detection of OsCERK1 protein accumulation level among different transgenic lines.

a, The OsCERK1-FLAG accumulation in UBQpro::OsCERK1-FLAG/UBQpro::OsCIE1-HA F2 sibling seedlings is equivalent to the UBQpro::OsCERK1-FLAG lines. The UBQpro::OsCERK1-FLAG/UBQpro::OsCIE1-HA double overexpression transgenic rice was generated by crossing. Then the separated F2 offspring seedlings from the same parent were used for analysis. The relative abundance is indicated under the bands. Actin detection serves as the loading control. b, Left: No significant difference of the transcript level of OsCERK1 was detected between UBQpro::OsCERK1-FLAG/proUBQ::OsCIE1-HA and UBQpro::OsCERK1-FLAG lines. CYCLOPHILIN2 was used as internal control for normalization. Right: No significant difference of the transcript level of OsCERK1 was detected between the F2 homozygous sibling plants expressing OsCERK1pro::OsCERK1-FLAG in the OsCIE1WT/Oscerk1-1 and homozygous Oscie1-1/Oscerk1-1 background. CYCLOPHILIN2 was used as internal control for normalization. Data were analyzed by two-tailed Student’s t-test (mean ± SD; n = 3, biologically independent samples). c, The OsCERK1-FLAG accumulation in OsCERK1pro::OsCERK1-FLAG/Oscerk1-1 and OsCERK1pro::OsCERK1-FLAG/ Oscerk1-1/Oscie1-1 background is comparable. The double mutant Oscerk1-1/Oscie1-1 was crossed with the transgenic Oscerk1-1 complementary line expressing OsCERK1 6,896 bp genomic DNA including its native promoter with 3×FLAG tags fused to the C terminal (Oscerk1COMWT) to generate F2 homozygous sibling plants expressing OsCERK1pro::OsCERK1-FLAG in the Oscerk1-1/OsCIE1WT (OsCERK1pro::OsCERK1-FLAG/Oscerk1-1) and Oscerk1-1/Oscie1-1 homozygous (OsCERK1pro::OsCERK1-FLAG/Oscerk1-1/Oscie1-1) background, respectively, for OsCERK1-FLAG protein accumulation studies. The relative abundance is indicated under the bands. Actin detection serves as the loading control. d, The stem strips of 10-day-old OsCERK1pro::OsCERK1-FLAG/Oscerk1-1 or OsCERK1pro::OsCERK1-FLAG/ Oscerk1-1/Oscie1-1 transgenic rice seedlings which are segregated from the same F1 parents were pre-treated with 300 μM the protein biosynthesis inhibitor cycloheximide (CHX) for 2 h then sampled at different time point in the presence or absence of chitin and/or the endomembrane trafficking inhibitor wortmannin (Wm). The gels of mock and chitin treatment samples were placed on the same membrane for transforming and exposure at the same time during the Western blot. Hsp82 detection serves as the loading control. e, Co-immunoprecipitation of HA-OsCEBiP and OsCERK1-FLAG transiently expressed in NIPB and Oscie1-1 rice protoplasts treated with or without 1 × 10−6 M chitin for 10 min. After chitin treatment, total protein was extracted and immunoprecipitation was performed with anti-HA agarose beads. Western blots were probed with FLAG and HA antibodies. f, Subcellular localization and internalization of OsCERK1-GFP was similar in Oscie1 mutant and wild-type. OsCERK1-GFP complementation lines within the Oscerk1-1 (OsCIE1) or Oscie1-1/Oscerk1-1 (Oscie1) mutant background were generated. The 5-day-old seedlings roots were pre-treated with the endocytic tracer FM4-64 (5 μg/mL) for 10 min, and then incubated with the endomembrane trafficking inhibitor BFA (100 µM) or mock for 30 min, followed by treatment of chitin (1 × 10−6 M) or mock for 30 min. The root samples were imaged with a laser scanning confocal microscope Leica SP8. OsCERK1-GFP primarily localized to the plasma membrane (PM) in roots of untreated or chitin-treated (30 min) plants. Treatment with BFA resulted in the formation of endomembrane aggregates in the OsCIE1 wild-type and Oscie1 mutant. The FM4-64 co-localized with the BFA-induced OsCERK1-GFP compartments. Scale bar, 10 µm. g, No significant changes of vesicle protein level of OsCERK1-FLAG was detected in Oscie1 mutant compared to wild-type. Plasma membrane fractions distinguished by the presence of the H+-ATPase marker and vesicle fractions labeled with the Arabidopsis trans-Golgi network/early endosome (TGN/EE) localized Qc-SNARE protein SYNTAXIN OF PLANTS61 (AtSYP61) were isolated. These fractions were prepared from protoplasts of OsCERK1-FLAG complementary rice seedlings under OsCIE1 or Oscie1 mutant backgrounds with mock or chitin treatment (for 30 min). All the protoplasts were transformed with AtSYP61-mCherry. Western blots probed with anti-FLAG, anti-mCherry or anti-H+-ATPase antibody. For a, c, the 10-day-old seedlings grown in axenic 1/2 MS culture were directly sampled without chitin treatment. Then the total protein was extracted and directly used for western blot with FLAG antibody. All the immunoblotting was individually conducted on separated gels in parallel and all the experiments above were repeated three times with similar results.

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Extended Data Fig. 8 OsCERK1 in vitro ubiquitination sites identified by mass spectrometry.

a, OsCERK1 was ubiquitinated by OsCIE1 at multiple lysine residues. Ubiquitinated lysine residues with a diglycine remnant identified by LC–MS/MS analysis were shown in red with amino-acid positions. b-k, MS/MS spectra of peptides containing ubiquitination residues of OsCERK1. b, Lys329; c, Lys347; d, Lys352; e, Lys445; f, Lys453; g, Lys458; h, Lys548; i, Lys558; j, Lys570; k, Lys584. MS spectra are outputs from the SEQUEST program. MS analysis was performed once. l, The structure of the OsCERK1 intracellular kinase domain was modeled by the online AlphaFold prediction software. Six lysines (Lys329, Lys347, Lys352, Lys445, Lys453, and Lys458) were localized within the core kinase region of OsCERK1. Specifically, Lys329 was positioned within the G loop, while Lys347 and Lys352 were situated in the αC helix and in proximity to the K-E salt bridge. Lys445 was found in the catalytic loop, and Lys453 and Lys458 were located within the activation segment.

Extended Data Fig. 9 OsCERK110KR construct can’t restore Oscerk1-1.

a, Individual lysine mutation does not block ubiquitination of OsCERK1 in vitro, whereas combined mutations of all ten lysines in OsCERK110KR largely blocked OsCIE1-mediated in vitro ubiquitination. Experiment was repeated three times with similar results. b, Mutation of all ubiquitinated 10 lysines blocks OsCERK1’s kinase activity. FLAG-tagged wild-type or mutant OsCERK1 was incubated with Myc-OsRLCK185K108E-His (an OsCERK1 substrate) in vitro. Anti-FLAG blotting was used to examine the abundance of OsCERK1 before the reaction (lower panel). After the reaction, Myc-OsRLCK185K108E-His was fractionated by SDS–PAGE containing Phos-tag acrylamide (upper panel) and normal SDS–PAGE (middle panel). c, No significant difference of the transcript level of OsCERK1 was detected among Oscerk1COMWTs and Oscerk1COM10KRs. Oscerk1COM10KRs were constructed by introducing 6,896 bp OsCERK110KR genomic DNA with 3×FLAG tags fused to its C terminal into Oscerk1-1 mutant. Data was analyzed by one-way ANOVA with Tukey’s test. Different letters indicate significant difference at P < 0.05. CYCLOPHILIN2 was used as internal control for normalization. Values are means ± SD (n = 3, biologically independent samples). d, No discernable difference of OsCERK1-FLAG protein accumulation was observed between Oscerk1-1/OsCERK1pro::OsCERK1-FLAG and Oscerk1-1/OsCERK1pro::OsCERK110KR-FLAG transgenic complementation plants. e, f, The chitin-induced MAPK activation and ROS burst were restored by OsCERK1 but not OsCERK10KR complementation. Stems of plants were treated with 1 × 10−6 M chitin for 10 min (e) and 1 h (f). For f, data was analyzed by one-way ANOVA with Tukey’s test. Different letters indicate statistical significance (P < 0.05). Values are means ± SD (n = 8, biologically independent samples). g, h, Ubiquitination of OsCERK1 inhibited its ATP hydrolysis activity. MBP-OsCERK1-His was affinity-purified with Ni-Sepharose. After elution with 250 mM imidazole and ultra-filtrated, loading of initially purified OsCERK1 is shown by CBB staining (bottom in h). Then OsCERK1 was incubated with OsCIE1 and ubiquitin (OsCERK1nUb) or without ubiquitin (OsCERK1WT) for ubiquitination for 2 h, and re-enriched by MBP amylose resins for secondly purification. After wash and elution with 50 mM maltose (loading is shown by CBB staining (up in h)), the kinase activity of OsCERK1WT or OsCERK1nUb was measured by detecting the production of ADP through a coupled NADH oxidation reaction that convert the ADP to ATP (g, left). ATP/protein/min of these proteins were calculated (g, right). Data were analyzed by paired two-tailed Student’s t-test (dash shows the means of three technical repeat signal ratio data (dots), *** means p value < 0.0001). i, OsCERK1pro::OsCERK1-FLAG/Oscerk1-1/Oscie1-1 seedlings grown in axenic culture displayed stronger basal MAPK activation level compared with the OsCERK1pro::OsCERK1-FLAG/Oscerk1-1 (Both lines were generated in Extended Data Fig. 7c). The 10-day-old seedlings grown in axenic 1/2 MS culture were directly sampled without chitin treatment. The total protein was extracted and directly used for western blot with p42/p44-MAPK antibody. All the immunoblotting were individually conducted on separated membranes in parallel and all the experiments above were repeated three times with similar results.

Source Data

Extended Data Fig. 10 OsCERK1-mediated phosphorylation inhibits OsCIE1’s ubiquitination activity.

a, 32P-γ-ATP kinase assays showing OsCERK1 directly phosphorylated OsCIE1. Protein loading control was determined with CBB staining. b-e, MS/MS spectra of peptides containing phosphorylated residues of OsCIE1. b, Thr206; c, Thr215; d, Thr216; e, Ser237. MS spectra were outputs from the SEQUEST program. f, The stronger phosphorylation tendency of OsCIE1 respond to 1 × 10−6 M chitin treatment for 10 min depends on OsCERK1 in rice protoplasts. The red triangle indicates the phosphorylated OsCIE1-FLAG. Relative protein abundance was indicated, and the relative phosphor-OsCIE1 signal ratio of chitin treatment to mock was calculated and illustrated in the right graph. Data was analyzed by paired two-tailed Student’s t-test (mean ± SD; n = 3, three independent repeat signal ratio data, Exact P values are provided.). g, Co-immunoprecipitation of OsCIE1-FLAG and OsCERK1-HA transiently expressed in rice protoplasts treated with or without 1 × 10−6 M chitin for 10 min. After chitin treatment, total protein was extracted and immunoprecipitation was performed with anti-FLAG agarose beads. Western blots were probed with FLAG and HA antibodies. h, Wild-type OsCIE1, an OsCIE1T206D, T215D, T216D, S237D (OsCIE14D) phosphor-mimetic version, and an OsCIE1T206A, T215A, T216A, S237A (OsCIE14A) phosphor-null version, interacted equally well with OsCERK1 in yeast two-hybrid assays. i, OsCIE1T206D, T215D, T216D, S237D (OsCIE14D), but not OsCIE1T206A, T215A, T216A, S237A (OsCIE14A) displayed dramatically reduced auto-ubiquitination (Anti-GST) and trans-ubiquitination ability (Anti-FLAG) to OsCERK1 in vitro. The reaction time was indicated. j, OsCERK1-mediated phosphorylation of OsCIE1 significantly impedes its ubiquitination function. The purified GST-OsCIE1, GST-OsCIE14A proteins were pre-incubated with MBP or MBP-OsCERK1ic for 1 h to acquire non-phosphorylated GST-OsCIE1 and phosphorylated GST-OsCIE1(pGST-OsCIE1). Then these GST-OsCIE1 varieties were repurified with glutathione resin to get GST-OsCIE1, GST-OsCIE14A, pGST-OsCIE1 and pGST-OsCIE14A. MBP-OsCERK1ic-FLAG was incubated with these proteins for ubiquitination for 30 min and 60 min. Western blots were probed with FLAG and HA antibodies. k, Protein accumulation level of OsCIE1 in complementary varieties lines. l, Complementary lines individually expressing OsCIE1 6,941 bp genomic DNA varieties including native promoter with 3×HA tags epitopes fused to the N terminal of OsCIE1 were generated (Oscie1-1COMWT, Oscie1-1COM4D, Oscie1-1COM4A) and employed for ROS burst assays. Stems of transgenic rice varieties were treated with 1 × 10−6 M chitin for 1 h. OsCIE14D couldn’t complement the Oscie1-1 mutant ROS burst phenotype as efficiently as wild-type, OsCIE14A. Values are mean relative light units (RLU) ± SD (n = 10 seedlings). For a, f, g, i, j, k all the blotting was individually conducted on separated membranes in parallel. For a, f, g, h, i, j, k, l experiments were repeated three times with similar results.

Source Data

Extended Data Fig. 11 Structure analysis of OsCIE1-U-box/OsUBC8 complex and Ser237 phosphorylation suppresses catalytic activity of OsCIE1.

a. The hydrophobic interactions on the dimeric interface of OsCIE1-U-box in the crystal structure of OsCIE1-U-box/OsUBC8. The electrostatic potential surface of RNAP was generated using APBS tools in Pymol. b. The polar interactions on the dimer interface of OsCIE1-U-box in the crystal structure of OsCIE1-U-box/OsUBC8. The two protomers are colored yellow and purple. Blue dashes, H-bond or salt-bridge interactions. c. The detailed interaction between OsCIE1-U-box and OsUBC8. Blue mesh, the simulated-annealing Fo-Fc difference map contoured at 2σ for the interface residues on the loop A of OsCIE1-U-box. d. The superimposition of dimeric OsCIE1-U-box (gray) in the crystal structure of OsCIE1-U-box/OsUBC8 and the 10 calculated conformers with the lowest energy of the wild-type OsCIE1-U-box NMR structure. e-n. 15N-edited HSQC-NOESY spectra (e-h, j-m) and 13C-edited HSQC-NOESY spectra (i and n) of wild-type OsCIE1-U-box (black) and its mutant U-boxS237D (blue). NOEs marked by pink squares were found between Ser237 and Cys234 in the wild-type OsCIE1-U-box (HNSer237-HNCys234, HβSer237-HNCys234 and HβSer237-HβCys234), while no corresponding NOEs were observed in the S237D mutant, which would contribute to the conformational differences in loop A between the two proteins. o, Yeast two-hybrid assay indicated that the wild-type OsCIE1 U-box domain and the phosphor-null U-boxS237A both can interact with OsUBC8 whereas the phosphor-mimetic U-boxS237D cannot. p, The Size-Exclusion Chromatography (SEC) showed that the OsCIE1 U-box domain formed a complex with OsUBC8 in vitro (the left graph), whereas OsCIE1S237D U-box did not (the right graph). q, The SEC fractions were analyzed by SDS-PAGE electrophoresis and visualized by Coomassie brilliant blue staining. r, Surface plasmon resonance (SPR) experiments were designed to evaluate the interaction between purified OsUBC8 and either immobilized OsCIE1-U-box (left) or OsCIE1-U-boxS237D (right) on the sensor chip. Ser237 phosphor-mimetic mutation inhibits the interaction between OsCIE1-U-box and OsUBC8. s, Phosphor-mimetic mutation S237D alone significantly inhibited the OsCIE1’s auto-ubiquitination activity (Anti-GST). Reaction time was indicated. t, Phosphor-mimetic mutation S237D alone significantly inhibited the OsCIE1’s trans-ubiquitination activity to OsCERK1 (Anti-FLAG). Reaction time was indicated. u, OsCIE14D phosphor-mimetic variant, but not OsCIE1T206D, T215D, T216D (OsCIE13D) displayed dramatically reduced auto-ubiquitination (Anti-GST) and OsCERK1 trans-ubiquitination (Anti-FLAG) activity. Reaction time was indicated. All the immunoblotting was individually conducted on separated membranes in parallel. All the experiments above were repeated three times with similar results.

Extended Data Fig. 12 OsCIE1 Ser237 is an evolutionally conserved switch controlling the interaction with E2.

a, OsCIE1 Ser237 is an evolutionarily conserved site for potential phosphorylation in other cross-kingdom species. The red overlined OsCIE1 sequence was used to generate anti-pSer237 antibodies. b, OsCERK1-FLAG was immunopurified from Oscerk1COMWT transgenic rice stems upon mock or chitin treatment for 10 min with FLAG beads. The bound protein was eluted by incubating with 3×FLAG peptide. This purified OsCERK1-FLAG was then incubated with GST-OsCIE1 or GST-OsCIE1S237A for in vitro kinase assay. Protein phosphorylation was detected by anti-S237p immunoblots. Purified OsCERK1-FLAG and recombinant GST-OsCIE1 variety proteins were detected by anti-FLAG antibody and stained with CBB, respectively. c, Ser237 mutation attenuates the phosphorylation level of OsCIE1 upon chitin treatment. Total proteins from Oscie1-1 mutant rice protoplasts expressing OsCIE1-FLAG varieties (OsCIE1 and OsCIE1S237A) were extracted and immunoprecipitated with FLAG beads. Lamda PPase (Phosphatase) treatment was regarded as the negative control. Immunoprecipitated protein was fractionated by SDS–PAGE containing Phos-tag acrylamide (upper panel) and normal SDS–PAGE (lower panel). d, Protein accumulation level of OsCIE1 in complementary varieties lines. e, The structural superimposition among crystal structures of OsCIE1-U-box/OsUBC8 structure (this study), DrCHIP-U-box/UbcH5a structure (PDB: 2OXQ), and HsCHIP-U-box/UBC13 (PDB: 2C2V). The insert shows the inward conformation of the conserved serine residue. f, Phosphor-mimetic DmCHIPS222D (Fruit fly), DrCHIPS220D (Zebrafish) and HsWDSUB1T413D (Human) illustrated dramatically impaired auto-ubiquitination activity after reaction for 2 h, while the phosphor-null DmCHIP mutation resembled the wild-type protein. GST-E3s were individually incubated with human E1, human E2 (UbcH5b), and HA tag ubiquitin (HA-Ub). Ubiquitinated proteins were detected by western blot using the anti-GST and anti-HA antibodies. g. Expression of OsCIE1 is induced upon 1 × 10−6 M chitin treatment for 2 h, which is dependent on OsCERK1. Data was analyzed by one-way ANOVA with Tukey’s test. Different letters indicate significant difference at P < 0.05. CYCLOPHILIN2 was used as internal control for normalization. Values are means ± SD (n = 3, biologically independent samples). h, A bistable switch model for the double negative feedback loop of OsCERK1 and ubiquitinated-OsCERK1. In the quiescent state, the activity balance between OsCIE1 and some unknown deubiquitinating enzymes (DUBs) ensure proper basal kinase activity levels of OsCERK1, eapecially during the early stage of chitin immunostimulation pathway. Perception of pathogenic microbe-derived chitin reduces the ubiqutination level and promotes the kinase activity of OsCERK1, which would in turn inhibit OsCIE1, and promote immune responses. However, the expression of OsCIE1 is upregulated during chitin stimulation, which will eventually lead to inhibition of OsCERK1 once the trigger (such as pathogen infection) is attenuated. For b-d and f, g, the experiment was repeated three times with similar results. All the mmunoblotting above was individually conducted on separated membranes in parallel.

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Supplementary Table 5

Crystal structure of OsUbox and UBC8 complex validation report

Supplementary Table 6

NMR strucutre of OsUboxWT validation report

Supplementary Table 7

NMR strucutre of OsUboxS237D validation report

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Wang, G., Chen, X., Yu, C. et al. Release of a ubiquitin brake activates OsCERK1-triggered immunity in rice. Nature (2024). https://doi.org/10.1038/s41586-024-07418-9

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